Heme Oxygenase-1 Modulates Human

Heme Oxygenase-1 Modulates Human
Respiratory Syncytial Virus Replication and

Lung Pathogenesis during Infection
This information is current as Janyra A. Espinoza, Miguel A. León, Pablo F. Céspedes,
of April 18, 2019. Roberto S. Gómez, Gisela Canedo-Marroquín, Sebastían A.
Riquelme, Francisco J. Salazar-Echegarai, Phillipe Blancou,
Thomas Simon, Ignacio Anegon, Margarita K. Lay, Pablo A.
González, Claudia A. Riedel, Susan M. Bueno and Alexis
M. Kalergis
J Immunol 2017; 199:212-223; Prepublished online 31 May
Downloaded from http://www.jimmunol.org/ by guest on April 18, 2019

2017;
doi: 10.4049/jimmunol.1601414
http://www.jimmunol.org/content/199/1/212
Supplementary http://www.jimmunol.org/content/suppl/2017/05/31/jimmunol.160141
Material 4.DCSupplemental
References This article cites 55 articles, 15 of which you can access for free at:
http://www.jimmunol.org/content/199/1/212.full#ref-list-1
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The Journal of Immunology
Heme Oxygenase-1 Modulates Human Respiratory Syncytial

Virus Replication and Lung Pathogenesis during Infection
Janyra A. Espinoza,* Miguel A. León,* Pablo F. Céspedes,* Roberto S. Gómez,*

Gisela Canedo-Marroquı́n,* Sebastı́an A. Riquelme,* Francisco J. Salazar-Echegarai,*
Phillipe Blancou,† Thomas Simon,† Ignacio Anegon,† Margarita K. Lay,‡
Pablo A. González,* Claudia A. Riedel,x Susan M. Bueno,* and Alexis M. Kalergis*,†,{
Human respiratory syncytial virus (hRSV) is the leading cause of severe lower respiratory tract infections in children. The devel-
opment of novel prophylactic and therapeutic antiviral drugs against hRSV is imperative to control the burden of disease in the
susceptible population. In this study, we examined the effects of inducing the activity of the host enzyme heme oxygenase-1 (HO-1) on
hRSV replication and pathogenesis on lung inflammation induced by this virus. Our results show that after hRSV infection, HO-1
induction with metalloporphyrin cobalt protoporphyrin IX significantly reduces the loss of body weight due to hRSV-induced dis-

ease. Further, HO-1 induction also decreased viral replication and lung inflammation, as evidenced by a reduced neutrophil in-
filtration into the airways, with diminished cytokine and chemokine production and reduced T cell function. Concomitantly, upon
cobalt protoporphyrin IX treatment, there is a significant upregulation in the production of IFN-a/b mRNAs in the lungs.
Furthermore, similar antiviral and protective effects occur by inducing the expression of human HO-1 in MHC class II+ cells
in transgenic mice. Finally, in vitro data suggest that HO-1 induction can modulate the susceptibility of cells, especially the airway
epithelial cells, to hRSV infection. The Journal of Immunology, 2017, 199: 212–223.
T
he human respiratory syncytial virus (hRSV) is the leading Epidemiological studies suggest that hRSV contributes to nearly
cause of lower respiratory tract illness (LRTI) in infants 33.8 million new episodes of LRTI in children ,5 y of age each
and children worldwide. hRSV produces reinfections year, with 3.4 million annual hospital admissions worldwide (3).
throughout life, generating frequent milder respiratory infections in Furthermore, hRSV infection is linked to neurologic symptoms in
adults as well as severe LRTIs in pediatric, elderly, and immu- patients, as well as learning impairment in animal models (4, 5)
nocompromised patients (1). LRTI can manifest as bronchiolitis or Currently, there are still no licensed vaccines nor specific antiviral
pneumonia, with the risk of death due to respiratory failure (2). drugs for the prophylaxis of hRSV disease in children and other sus-
ceptible populations (6). The only approved therapeutic approaches
are palivizumab (7), a humanized mAb that protects against hRSV
*Instituto Milenio en Inmunologı́a e Inmunoterapia, Departamento de Genética Mo-
lecular y Microbiologı́a, Facultad de Ciencias Biológicas, Pontificia Universidad
infection in high-risk infants, and ribavirin, an antiviral nucleoside
Católica de Chile, Santiago 8331150, Chile; †Centre de Recherche en Transplantation analogue that is rarely used due to toxicity concerns and question-
et Immunologie UMR1064, INSERM, Université de Nantes, Nantes 44093, France; able benefits (7). For these reasons, intense research has focused on
‡
Departamento de Biotecnologı́a, Facultad de Ciencias del Mar y Recursos Biológ-
icos, Universidad de Antofagasta, Antofagasta 1270300, Chile; xInstituto Milenio en finding novel vaccines or antiviral agents to prevent hRSV infection.
Inmunologı́a e Inmunoterapia, Departamento de Ciencias Biológicas, Facultad de During hRSV disease, epithelial cells in the distal airway re-
Ciencias Biológicas y Facultad de Medicina, Universidad Andrés Bello, Santiago
8370134, Chile; and {Departamento de Endocrinologı́a, Facultad de Medicina, Pon-
spond to viral infection by secreting proinflammatory cytokines
tificia Universidad Católica de Chile, Santiago 8331150, Chile and chemokines that promote an exacerbated recruitment of in-
ORCIDs: 0000-0002-7647-5454 (M.A.L.); 0000-0001-6709-699X (F.J.S.-E.); 0000- filtrating cells, mainly neutrophils, leading to inflammation and
0002-4081-7977 (M.K.L.); 0000-0001-7709-6870 (P.A.G.). tissue damage (8). Therefore, the design of new strategies to
Received for publication August 15, 2016. Accepted for publication April 24, 2017. prevent hRSV diseases must consider parameters such as the in-
This work was supported by grants from Comisión Nacional de Investigación Cien- hibition of viral replication and reduction of lung inflammation.
tı́fica y Tecnológica/Fondo Nacional de Desarrollo Cientı́fico y Tecnológico (Post- Heme oxygenase-1 (HO-1) is a metabolic enzyme that catalyzes
doctorado 3140455, 1140011 and 1150862), Instituto Milenio en Inmunologı́a e
Inmunoterapia (P09-016-F). J.A.E. and R.S.G. are Comisión Nacional de Investiga- the degradation of heme into carbon monoxide, biliverdin, and free
ción Cientı́fica y Tecnológica de Chile Fellows. iron (9). This enzyme has anti-inflammatory and antioxidant
Address correspondence and reprint requests to Dr. Alexis M. Kalergis, Instituto properties, which modulate host innate and adaptive immune re-
Milenio en Inmunologı́a e Inmunoterapia, Departamento de Genética Molecular y sponses (10). The immunomodulatory capacity of HO-1 has been
Microbiologı́a, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de
Chile, Av. Portugal 49, Alameda 340, Santiago 8331010, Chile. E-mail address: demonstrated in several models, such as the LPS-induced acute
akalergis@bio.puc.cl lung inflammation in which HO-1 activation decreased the mi-
The online version of this article contains supplemental material. gration of polymorphonuclear leukocytes to the lung, reducing
Abbreviations used in this article: BALF, bronchoalveolar lavage fluid; CoPP, cobalt oxidative tissue damage (11). Furthermore, the pharmacological
protoporphyrin IX; DD Ct, DD threshold cycle; DC, dendritic cell; DOX, doxycycline; induction of HO-1 inhibits dendritic cell (DC) activation and
HCV, hepatitis C virus; HEp-2, human laryngeal epidermoid carcinoma number 2; HO-
1, heme oxygenase-1; hRSV, human respiratory syncytial virus; LM, littermate; LRTI, immunogenicity (12), suppressing cytokine secretion and the ca-
lower respiratory tract illness; MHC-II, MHC class II; MHC-II2, MHC-II negative; pacity to prime T cells (13). Also, recent studies have shown that
MHC-II+, MHC class II positive; MOI, multiplicity of infection; N, nucleoprotein; HO-1 can display important antiviral properties. Specifically, up-
qPCR, quantitative PCR; SnPP, tin protoporphyrin IX dichloride; UT, untreated.
regulation of HO-1 was shown to diminish infection by several
Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 viruses, including Ebola, influenza, enterovirus, hepatitis C virus
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601414
The Journal of Immunology 213
(HCV), hepatitis B virus, and HIV. During infection by these vehicle (NaOH diluted in media), or 50 mM CoPP (HO-1 inducer)
viruses, HO-1 induction protects infected tissues, such as the liver (Frontier Scientific), or 50 mM SnPP (HO-1 inhibitor) (Frontier Scientific),
and incubated at 37˚C in 5% CO2 (19). After 2 h, supernatants were re-
and lungs, from virus-induced oxidative injury (10). Although the moved, and cells were inoculated with infectious hRSV at a multiplicity of
mechanisms of action for HO-1 during viral infection have not infection (MOI) equal to 1 PFU per cell and incubated for 2 h in OptiMem I
been elucidated, available data suggest a direct effect on virus Reduced Serum Medium (preinfection). As a control, cells were inoculated
proteins or on the activation of cellular processes that interfere either with the same media (untreated [UT]) or with mock (supernatant of
with virus replication, such as the type I IFN response (14). uninfected HEp-2 or with ultraviolet-inactivated hRSV). Each treatment was
also performed during the hRSV infection for 24 h (postinfection). In both
The HO-1 expression can be highly induced by analogs of heme, cases, after 24 h replication was determined by measuring the nucleoprotein
such as hemin and other metalloporphyrins. The best-characterized (N) RNA, and copies were quantified by real-time quantitative PCR (qPCR).
inductor of HO-1 is cobalt protoporphyrin (CoPP), which promotes In addition, supernatants from infected cells for each treatment were col-
the upregulation of HO-1 gene expression (15). Thus, the primary lected at 48 h postinfection to determine viral titer by immuno-plaque assay.
mechanism involved in the upregulation of the HO-1 enzyme Viral immuno-plaque assay
seems to be the enhancement of gene transcription (15, 16). Al-
losteric HO-1 inhibitors can also induce upregulation of HO-1 The viral titer in supernatents of bronchoalveolar lavage fluid (BALF) was
determined by immunocytochemistry. Briefly, infectious supernatants were
expression, as occurs with tin protoporphyrin IX dichloride serially diluted (10-fold dilutions), added to 96-well plates with HEp-2
(SnPP) (17). However, despite inducing HO-1 expression, SnPP monolayers (80% confluence), and incubated for 48 h at 37˚C. Later,
irreversibly inhibits the activity of this enzyme (18). cells were fixed with 2% paraformaldehyde-PBS and permeabilized
Taking into consideration the multifunctional properties of HO-1, 20 min with 0.2% saponin-PBS. Intracellular staining was performed with
an anti-N-hRSV (clone 1E9/D1) Ab for 1 h (dilution 1:500, 0.2% saponin-
in this study we examined the effects of HO-1 induction on the path- PBS). Cells were washed twice and incubated with anti-mouse IgG-HRP
ogenesis caused by hRSV in vitro and a mouse infection model.

(dilution 1:200; Invitrogen, Molecular Probes) for 45 min. After washing
Specifically, we assessed the modulatory effects of HO-1 on airway the complex twice, the substrate TRUE-BLUE peroxidase (KPL) was
epithelial cells infected with hRSV. Additionally, we evaluated whether added to cells and incubated for 10 min at room temperature.
either CoPP or transgenic induction of HO-1 displayed antiviral and Quantitative hRSV by ELISA
anti-inflammatory effects in the airways of hRSV-infected mice.
Briefly, 100 ml of virus-containing supernatants from hRSV-infected A549
cells at an MOI of 1, and treated with CoPP, SnPP, or vehicle (6–100 mM)
Materials and Methods were used to coat ELISA plates for 2 h at 37˚C. Then, plates were blocked
Mice and hRSV production for 2 h with PBS containing 10% of FBS. After washing three times with
PBS-tween 20 0.01%, goat anti-hRSV-HRP conjugated (Abcam) diluted in
C57BL/6J and BALB/cJ wild-type mice were obtained from The Jackson 1:500 in PBS FBS 10% was added to the wells for 2 h. Finally, after
Laboratory for the generation of pIi-TTA-TetO-HO-1 transgenic mice. PIi- washing five times with PBS-tween 20 0.01%, bound Ab was detected by
TTA mice were a kind gift from Christophe Benoist (19). TetO-HO-1 mice, addition of tetramethylbenzidine peroxidase substrate (BD), stopped with
located upstream of the cDNA sequence and located downstream of the 1 M H2SO4, and analyzed at 450 nm by an ELISA Plate Reader.
cDNA, was cloned at the Not-I/Xho-I sites into the pBluKSM-tet-O-CMV
vector containing the followed by the human b-globin intron and the bo- Quantification of hRSV binding by flow cytometry and
vine growth hormone polyA. Western blotting
TetO-HO-1 transgenic mice were generated by pronuclear microinjection of
CBA/C57BL6 eggs with a DNA fragment containing a Tet-responsive element HEp-2 cells were detached using versene solution (Sigma-Aldrich), washed,
downstream a minimal CMV promoter, the human b-globin intron, the human exposed to 50 mM of CoPP, SnPP, or vehicle, and chilled on ice for 30 min.
HO-1 cDNA, and the bovine growth hormone polyA. All mice were main- Cells were washed and exposed to hRSV (MOI = 1) concomitantly with
tained at the pathogen-free facility of the Pontificia Universidad católica de CoPP, SnPP, or vehicle for 1 h at 4˚C. For flow cytometry analyses, cells were
Chile and manipulated according to guidelines approved by the institution’s washed, fixed (4% paraformaldehyde), and anti–hRSV F-protein FITC–
Bioethical Committee. hRSV serogroup A2, strain 13018–8, is a clinical conjugated Ab (1:200, ab20391; Abcam) was added and incubated for 1 h at
isolate obtained from the Instituto de Salud Pública de Chile. In most ex- 4˚C. For Western blotting analyses, hRSV- exposed cells were washed,
periments, a mock control was included consisting of supernatants collected resuspended in RIPA buffer, and incubated at 95˚C for 10 min. SDS-PAGE,
from uninfected human laryngeal epidermoid carcinoma number 2 (HEp-2) on 10% Bis/Tris gels and in MES buffer (Invitrogen), was performed and the
cells kept in culture for the same period of time as infected cells. indicated protein was transferred to nitrocellulose membranes. The mem-
branes were then exposed to anti-N protein Ab, anti HO-1 (ab13248; Abcam),
Virus preparation or anti–b actin (6221102; BioLegend), followed by HRP-labeled secondary
Ab (goat anti-mouse; Invitrogen, Molecular Probes). Chemiluminescence
HEp-2 cells (CCL-23; American Type Culture Collection) were used to
(Amersham ECL Prime Western blotting Detection Reagent; GE Healthcare,
propagate hRSV serogroup A2, strain 13018–8 (clinical isolate obtained
Little Chalfont, U.K.) was detected using a thermal imaging system.
from the Instituto de Salud Pública de Chile), as previously described (4).
Briefly, HEp-2 cell monolayers were grown in T75 flasks with DMEM DC infection and pharmacological modulation of HO-1
(Life Technologies, Invitrogen, Carlsbad, CA) supplemented with 10%
FBS. Flasks containing 5 ml of culture medium were inoculated with 2 3 105 Bone marrow–differentiated DCs from C57BL/6 mice were prepared as
PFU of hRSV and incubated at 37˚C. After viral adsorption (3 h), super- previously described (20, 21). Analyses for expression of surface markers
natants were replaced with fresh medium (DMEM 1% FBS) and incubated by flow cytometry revealed a typical phenotypic profile for immature DCs
for 48 h or until the visible cytopathic effect was observed. Cells were (.75% of CD11c+ cells). On day 5 of culture, DCs were inoculated with
harvested, the flask content was pooled, then spun twice at 300 3 g for 10 hRSV or ultraviolet-hRSV for 2 h at an MOI equal to 1 PFU per cell. As
min to remove cell debris. In parallel, supernatants of noninfected HEp-2 controls, DCs were left UT or inoculated with similar volumes of super-
monolayers were collected as previously described, and used as noninfec- natants from uninfected HEp-2 cultures (mock). Then 48 h postinfection,
tious control (mock). Viral titer of supernatants was determined by immu- the viability of DCs was determined by trypan blue exclusion. In contrast,
nohistochemistry. Ultraviolet-inactivated hRSV (ultraviolet-hRSV) was immature DCs (1 3 106 cells) were pulsed for 2 h with 50 mM CoPP, SnPP,
obtained exposing ice-packed virus preparation vials for 45–60 min at or vehicle control. Cells were then washed twice and cultured 4 h before
302 nm using a 15 W lamp trans-illuminator. hRSV inoculation. Then, HO-1 mRNA and protein levels were analyzed by
qPCR and flow cytometry or immunofluorescence, respectively.
hRSV infection of A549 cells and pharmacological modulation
of HO-1 expression Pharmacological modulation of HO-1 expression and hRSV
challenge in vivo
Human alveolar type II–like pulmonary epithelial cells (A549 cells)
(kindly provided by Dr. P. Piedra, Baylor College of Medicine) were Male 6–8 wk old BALB/cJ wild-type mice were pretreated i.p. (7.6 mmol/kg)
maintained in DMEM medium containing 10% (v/v) FBS, 100 IU/ml either with CoPP to induce the HO-1 expression, or with SnPP to inhibit the
penicillin, and 100 mg/ml streptomycin. A549 cells were treated with activity of HO-1 as previously described (22), 24 h before viral challenge.
214 HO-1 AS A NEW PROPHYLACTIC TREATMENT AGAINST hRSV INFECTION
Mice, treated with NaOH (diluted in PBS), were included as the vehicle light-inactivated hRSV, and mock. DCs were then stained with anti-CD11c-
control. After 24 hr mice were anesthetized with ketamine or xylazine (80 and PE-Cy7 (clone HL3; BD Pharmingen), anti-IA/IE-PerCP-Cy7 (clone M5;
8 mg per kg, respectively) and challenged intranasally with either 1 3 106 PFU BD Pharmingen), and anti-hRSV N-AF647 conjugated (clone 1E9/D1) in
of hRSV or an equal volume of mock (as non-infectious control). Animal body 10% of FBS in PBS as a blocking solution. For HO-1 intracellular staining,
weight was recorded daily postinfection. At day 4 postinfection, mice were fixed cells were incubated with anti-mouse HO-1 mAb (Abcam) in per-
terminally anesthetized by i.p. injection with a mixture of ketamine and meabilization buffer (1% saponin, 10% FBS in PBS) for 45 min at 4˚C.
xylazine. BALF and lung tissue samples were collected for further analyses. Then, cells were washed and stained with goat anti-mouse IgG-AF488
(Invitrogen). For cell infiltration analysis, lung samples were homoge-
Infection of rtTA-HO-1 transgenic mice nized and filtered using a 40 mm cell strainer. BALF was centrifuged
Female or male 6–8 wk old rtTA-HO-1 mice and littermate mice were at 300 3 g for 5 min, washed, and stained with anti-CD11b-APC (clone
treated with 800 mg/ml doxycycline (DOX) and 36 mg/ml sucrose in CBRM1.5; BD Pharmingen), anti-CD11c-PE (clone CBRM1.5; BD
drinking water, protected from the light, to induce the expression of human Pharmingen), anti-IA/IE-APC-Cy7 (clone M5; BD Pharmingen), anti-Ly6C-
HO-1 in MHC class II positive (MHC-II+) cells. Then 48 h later, mice were PercCP 5.5 (clone AL-21; BD Pharmingen), and anti-Ly6G-FITC (clone
anesthetized with ketamine/xylazine (20 and 1 mg/kg, respectively) and RB6-8C5; BD Pharmingen). In addition, lung samples were stained for HO-1
challenged intranasally with either 1 3 106 PFU of hRSV or an equal mAb, as described above. For the A549 cell line, cells were stained with anti
volume of mock (as non-infectious control). Animal body weight was hRSV F-Alexa Fluor 647, conjugated, and washed for further analysis. All
recorded daily postinfection. At day 4 postinfection, mice were terminally samples were acquired on a FACS Canto II flow cytometer (BD Biosciences,
anesthetized by i.p. injection with a mixture of ketamine and xylazine. San Jose, CA) and analyzed using FlowJo 7.6 software.
BALF and lung tissue samples were collected for further analyses. Lung histopathology analyses
Genotyping of rTA-HO-1 To perform histopathology analyses without losing significant tissue ar-
Mice were bled from the cheek into 100 ml of heparin (125 UI/ml). For chitecture, before BALF collection, the major bronchus of the left lung was
DNA purification, we used the DNeasy Blood and Tissue Kit following the clamped using 10 cm Kelly hemostatic forceps. After BALF of the right
lung, the left lung was fixed with 4% paraformaldehyde, then paraffin

manufacturer’s instructions. Consequently, we performed a PCR for each
transgene inserted in the transgenic mice: rtTA and tHO-1 using GoTaq G2 embedded using a Leica ASP300S enclosed, automatic tissue processor
Flexi DNA Polymerase (Promega). Then, samples were loaded in an (Leica Microsystems, Wetzlar, Germany). Then, 4 mm-thick tissue sections
agarose electrophoresis. The mice that only had the tHO-1 gene, but lacked were obtained using a Microm HM 325 Rotary Microtome (Thermo Sci-
the tTA gene were named littermate (LM), which were used as controls. entific), before being mounted and stained for histopathology analyses
using H&E.
Quantitative real time RT-PCR
Cytokines and chemokines measured by ELISA
Total RNA was isolated from tissues or cell cultures by using the Trizol reagent
(Life Technologies, Invitrogen), according to the manufacturer’s instructions. Immunoreactive CXCL1/KC were quantified by using a double Ab ELISA
cDNA synthesis from total RNAs was performed using the ImProm-II Re- kit (DuoSet; R&D Systems, Minneapolis, MN). IL-6, IL-4, IL-10, and
verse Transcription kit (Promega) and random primers. qRT-PCR reactions IFN-g were quantified by using Ab ELISA kit (OptEIA; BD Pharmingen),
were carried out using a StepOne plus thermocycler (Applied Biosystems). and CCL3/MIP-1a detection was performed following the manufacturer’s
The abundance of HO-1 and Nrf2 mRNAs was determined by relative ex- protocol (Ready Set Go!; Affymetrix).
pression to the respective housekeeping gene by the 2-DD threshold cycle Measurements of T cell function during hRSV infection
(DDCt) method. For N-gene expression, absolute quantification data were
expressed as the number of hRSV N-gene copies for each 5 3 103 copies Lymph nodes from infected and mock BALB/cJ mice or tTA-HO-1 con-
of b-actin transcript, as previously described (4). The following primers were ditional transgenic mice of each treatment were removed and mechanically
used: hRSV N Forward 59-GCTAGTGTGCAAGCAGAAATC-39 and homogenized in 1 3 PBS. After erythrocyte lysis with ACK buffer
Reverse 59-TGGAGAAGTGAGGAAATTGAGTC-39, mouse HO-1 59- (150 mM NH4CL, 10 mM KHCO3, 0.15 mM EDTA), cells were resus-
CCTCTGACGAAGTGACGCC-39 and Reverse 59-CAGCCCCACCA- pended at a final concentration equal to 5 3 106 cells per ml in RPMI
AGTTCAAA-39, human HO-1 Forward 59-AGGCAGAGGGTGATA- 1640 medium, supplemented with 10% FBS, 1 mM nonessential amino
GAAGAGG-39 and Reverse 59-TGGGAGCGGGTGTTGAGT-39, mouse Nrf-2 acids, 2 mM glutamine, 1 mM pyruvate, 10 mg/ml penicillin G, 100 mg/ml
Forward 59-TTC TTT CAG CAG CAT CCT CTC CAG-39 and Reverse streptomycin, 50 mg/ml gentamicin, and 50 mM 2-ME. Then, single-
59-ACA GCC TTC AAT AGT CCC GTC CAG-39, mouse IFN-a Forward cell suspensions were left untreated or stimulated with ultraviolet-
59-TCC TGA ACC TCT TCA CAT CAA A-39 and Reverse 59-ACA GGC hRSV or anti-CD3ε/CD28. After 72 h of incubation (37˚C, 5% CO2),
TTG CAG GTC ATT GAG-39, mouse IFN-b Forward 59-AGC TCC AAG culture supernatants were analyzed for IFN-g production by sandwich
AAA GGA CGA ACA-39 and Reverse 59-GCC CTG TAG GTG AGG TTG ELISA, and the expression of activation marker CD69 was measured
AT-39, mouse b-actin Forward 59-ACCTTCTACAATGAGCTGCG-39 and on the surface of cells by flow cytometry (FACS Canto II flow
Reverse 59-CTGGATGGCTACGTACATGG-39. cytometer [BD Biosciences, San Jose, CA] and analyzed using FlowJo
7.6 software).
Laser confocal microscopy
DC maturation and APC assay
DCs (1 3 106 cells) were produced as described above. Briefly, DCs were
grown over 12 mm microscope cover glasses (Marienfeld-Superior, DE). Immature DCs from conditional transgenic mice were incubated for 2 h
Then, DCs were inoculated, as mentioned above, with hRSV, ultraviolet-hRSV with 50 mM CoPP or 1.5 mg/ml DOX (for all the incubation period).
or mock, and cultured for 48 h at 37˚C. Inoculated DCs were prepared for Then, cells were washed once and incubated with 700 ml of fresh RPMI
confocal microscopy analysis, as previously described (23). Briefly, DCs were 1640 medium. Maturation of DCs was induced by LPS treatment (1 mg/ml)
washed and fixed with 2% p-formaldehyde for 15 min at 4˚C. Then, cells were for 16 h (Escherichia coli 0111; Invitrogen) and the surface expression of
permeabilized with 0.05% saponin-PBS for 15 min at 4˚C. Next, cover glasses CD40, CD86, and CD80 molecules was measured by flow cytometry.
were passed to a cold chamber and DCs were double-stained with 1/200 Also, cells were pulsed with 50 mg/ml of OVA protein, non-OVA pulsed
anti-mouse HO-1 mAb (Abcam), already dissolved in 0.05% saponin-PBS. cells 6 LPS were used as a control for antigenic presentation. After
These preparations were incubated overnight at 4˚C in darkness. The next day, overnight incubation, cells were washed twice, detached, counted, and
cells were washed with PBS and stained with 1/200 goat anti-mouse IgG- plated in round-bottom 96-well plates. Then, 50,000 DCs were incubated
Alexa Fluor 488 (Invitrogen) and 1/200 goat-anti rabbit IgG-AF555 (Invi- with 100,000 purified (over 90%; Miltenyi Biotec) OVA-specific TCR
trogen) secondary Abs, and incubated for 3 h at 4˚C in darkness. After OT-II T CD4+ cells (relationship 2:1 T cells:DCs). After 48 h of cocul-
washing the complex, cells were dried and mounted with DABCO mounting ture, supernatants were collected for IFN-g secretion, and T cells were
medium for confocal microscopy. Fluorescence measurements were per- stained for CD69 activation marker.
formed on a FluoView FV1000 confocal microscope (Olympus). After image
recording (at 403 magnification), each channel (HO-1, nuclei, and transmis- Statistical analyses
sion) was analyzed separately using Olympus Fluoview version 3.0 software.
All statistical analyses were performed using GraphPad Prism Software
Flow cytometry version 6.1. Statistical significance was assessed using the Student t test and
one-way ANOVA test with a posteriori Bonferroni test or Kruskal–Wallis
DCs, A549, and HEp-2 cells were inoculated, as mentioned above, and ANOVA test with a posteriori Mann–Whitney U test. Differences were
cultured for 24 or 48 h, respectively, in the presence of hRSV, ultraviolet considered significant when p , 0.05.
Results harvested 48 h postinfection, and infective virus particle produc-

HO-1 induction inhibits hRSV replication and virus particle tion was measured by detection of the hRSV F protein by ELISA.
production in vitro We observed that production of virus particles in supernatants was
inversely correlated with increasing CoPP doses postinfection in
Upregulation of HO-1 suppresses infection of several viruses,
including HIV, HCV, hepatitis B virus, Ebola virus, and influenza A549 cells (Fig. 1A, white circles). Conversely, treatment with
virus (16). To evaluate whether HO-1 induction reduces hRSV increasing doses of SnPP was directly associated with hRSV
infection in vitro, we pretreated the human airway epithelial cell production in the supernatant of the treated A549 cells (Fig. 1A,
line A549 with increasing concentrations of CoPP (an HO-1 in- black squares). Because treatment with 50 mM of CoPP resulted in
ducer), vehicle, or SnPP (an allosteric HO-1 inhibitor) for 2 h more than a 60% reduction in hRSV production, as compared with
(Fig. 1A, 24). Supernatants of cells were then removed and cells cells treated with vehicle, we selected this concentration for fur-
were washed with fresh medium and infected with hRSV at an ther experiments. Moreover, to address whether HO-1 upregula-
MOI equal to 1 for 2 h. After viral adsorption, cells were washed tion affects the generation of the viable progeny of hRSV viral
with medium to remove unbound virus. For postinfection treat- particles, supernatants from infected A549 cells treated with
ment, cells were infected as described above, and CoPP was added CoPP, vehicle, or SnPP were harvested to quantify virus titers by
to the cells and incubated for 24 or 48 h. Induction of HO-1 by immune-plaque assays. We found that hRSV-infected A549 cells
CoPP was assessed by flow cytometry and Western blotting treated with 50 mM of CoPP postinfection showed reduced virus
(Fig. 1D, Supplemental Fig. 1), respectively. Supernatants were titers by 3 log units as compared with cells treated with vehicle
FIGURE 1. HO-1 induction reduces hRSV replication in human A549 cells. A549 cells were infected with hRSV at MOI 1 in the presence or absence of
CoPP (HO-1 inducer), SnPP (HO-1 inhibitor) or vehicle control. (A) Dose-response curves measured by ELISA to detect hRSV F protein in supernatants
from infected A549 cells treated with CoPP (white circles, 450 nm), SnPP (black squares), or vehicle (white diamonds). (B) Viral titers of supernatants from
infected cells treated with vehicle, CoPP, or SnPP 48 h postinfection with hRSV. (C) Copy number for hRSV-N RNA in infected A549 cells per ng of cDNA
at either 24 h postinfection for both cells, preinfection treatment with drugs (white bars), or treatment with drugs postinfection (black bars). (D) Repre-
sentative Western blotting from total protein homogenates for HO-1 (upper panel), hRSV-N (middle panel), and b-actin (lower panel), in HEp-2 cells after
2 h of viral absorption at 4˚C to assess viral binding, in the presence of vehicle, CoPP, or SnPP. (E) Overlaid histograms of flow cytometry analyses for
surface hRSV F protein expression in HEp-2 cells after 2 h of viral absorption at 4˚C to assess viral binding, in the presence of vehicle, CoPP, or SnPP. (F)
Mean fluorescence intensity of surface hRSV F protein–expressing cells after 2 h of viral absorption at 4˚C, to assess viral binding in the presence of
vehicle, CoPP or SnPP. (G) Overlaid histograms of flow cytometry analyses for surface hRSV F protein expression in HEp-2 cells after 2 h of viral ab-
sorption at 4˚C and 5 h of incubation at 37˚C, to assess viral entry in the presence of vehicle, CoPP, or SnPP. (H) Mean fluorescence intensity of surface
hRSV F protein–expressing cells after 2 h of viral absorption at 4˚C and 5 h of incubation, to assess viral entry, in the presence of vehicle, CoPP, or SnPP.
Data shown are mean 6 SEM from three independent experiments. Data were analyzed by one-way ANOVA and Bonferroni posttest (*p , 0.05, **p ,
0.001, ***p , 0.0001).
(Fig. 1B). Furthermore, because N transcription can be considered HO-1 was expressed in DCs upon hRSV infection. Bone marrow–
a measurement of hRSV viral replication within infected cells derived DCs were exposed to hRSV, ultraviolet-hRSV, or mock.
(25), we evaluated this transcript in cells treated as indicated HO-1 expression was determined by qPCR, flow cytometry, and
above. We observed a significant reduction in viral N mRNA immunofluorescence. As shown in Fig. 2A, a significant increase
amounts in cells treated with CoPP, both added preinfection and in HO-1 mRNA expression levels was observed in hRSV-infected
postinfection (Fig. 1C). Conversely, treatment with SnPP in- DCs, as compared with mock or untreated cells at 48 h postin-
creased the amounts of hRSV N-transcripts in infected cells. fection. On the contrary, ultraviolet-hRSV–inoculated DCs did not
Equivalent to A549, virus particle production and viral replication show an upregulation of HO-1 mRNA levels, suggesting that
were similarly influenced by the HO-1 expression in HEp-2 cells HO-1 induction depends on hRSV gene transcription. Consistent
(data not shown). In addition, to address the stage of the hRSV with this observation, flow cytometry analyses exhibited a sig-
replication cycle that was affected by HO-1, we evaluated the nificant increase in the expression of HO-1 in hRSV-inoculated
effects of HO-1 induction, mediated by CoPP, on hRSV binding DCs (Fig. 2B). Confocal microscopy experiments further con-
and entry. Previous reports have described that the active fusion firmed these findings, showing significantly higher HO-1 levels in
process of hRSV is inhibited in HEp-2 epithelial cells at 4˚C, hRSV-infected DCs, as compared with control cells (Fig. 2C). The
whereas viral binding still occurs (26, 27). Viral binding was HO-1 expression levels seen in hRSV-infected DCs were equiv-
assessed by Western blotting, measuring the hRSV-N protein alent to the HO-1 levels induced by CoPP (Fig. 2C), suggesting
over total protein collected from these cells (Fig. 1D), as well as that hRSV is a potent stimulus for HO-1 expression in DCs. Of
by flow cytometry analyses of surface expression of the hRSV note, the subcellular location of HO-1 was mainly situated near
F-protein (Fig. 1E, 1F). Interestingly, no significant changes were the plasma membrane in CoPP-treated DCs, but displayed a ho-
observed for the expression of hRSV-N (Fig. 1D, middle panel) mogeneous distribution throughout the cytoplasm and plasma

nor the surface expression of the hRSV-F protein (Fig. 1E, 1F), membrane in hRSV-infected DCs, suggesting that the differential
suggesting that HO-1 induction has no effect on hRSV binding. To distribution of HO-1 could be associated with different functions.
evaluate viral entry, cells were infected at 4˚C for 1 h, as described
HO-1 induction limits viral replication in vivo and provides
above, washed and treated with CoPP, vehicle, or SnPP. Cells were
protection against hRSV infection in mice
then incubated at 37˚C for 5 h. Viral entry was measured by detecting
intracellular expression of hRSV-F protein by flow cytometry. When Considering hRSV infection was significantly reduced in vitro as a
CoPP-treated cells were compared with vehicle-treated or SnPP- result of HO-1 induction in airway epithelial cells and that this
treated cells, no significant differences were observed (Fig. 1G, enzyme can display a potent anti-inflammatory activity (30–32),
1H). Virus particle production and viral replication in HEp-2 cells we evaluated whether the pharmacological induction of HO-1
were affected similarly as for A549 cells (data not shown). in vivo can modulate hRSV infection in mice. Thus, BALB/cJ
Thus, these results suggest that although HO-1 induction neg- mice were treated either with CoPP or SnPP 24 h before hRSV
atively modulates hRSV replication in human alveolar epithelial infection. Then, animals were challenged intranasally with hRSV
cells, the inhibition of HO-1 activity promotes replication of this (1 3 106 PFU) or with equivalent volumes of a mock solution (as
virus in these cells. However, HO-1 induction does not affect a non-infectious control). Changes in body weight were monitored
hRSV binding nor fusion in infected cells. as a parameter of disease progression for 7 d postinfection (4). As
shown in Fig. 3A, a noticeable weight loss was observed after
hRSV infection increases HO-1 expression in DCs hRSV infection in vehicle-treated mice. Remarkably, CoPP-
Because HO-1 can modulate the function of APCs (12, 28) and treated mice displayed accelerated kinetics of body weight re-
previous reports have shown that hRSV infects DCs, impairing covery after hRSV infection, as compared with vehicle-treated
their capacity to prime T cells (14, 29), we evaluated whether mice (Fig. 3A). Consistently, significant differences between
FIGURE 2. hRSV infection induces HO-1

expression in DCs. DCs were incubated with
mock, ultraviolet-hRSV or hRSV at MOI 1,
CoPP was used as a control of HO-1 induction
and SnPP as an inhibitor of HO-1 activity, then
cells were analyzed after 48 h. (A) HO-1 mRNA
levels quantified by qPCR. Expression of rela-
tive mRNAs for target genes was normalized to
b-actin levels using the 2-DDCt method (UT cells
were used as a reference control) (***p , 0.0001,
*p , 0.0154; one-way ANOVA, Bonferroni
posttest). (B) Mean fluorescence intensity (MFI)
for HO-1 in MHC-II+ cells for each treatment
(*p , 0.05, ns, p , 0.0575; one-way ANOVA,
Bonferroni posttest). (C) Confocal microscopy
images show merged channels for HO-1 expres-
sion (green fluorescence) in DC cytoplasm of
hRSV-infected and CoPP-treated DCs. In addi-
tion, the nuclei are shown in blue. Data shown are
mean 6 SEM from three independent experi-
ments. **p , 0.005; one-way ANOVA, Bonfer-
roni posttest.
CoPP-treated and vehicle-treated mice were observed at days 2, 3, infiltration were consistent with previous data (21), showing that
and 4 postinfection with hRSV. In agreement with the body weight hRSV challenge caused significant infiltration of inflammatory
recovery data, a significant reduction of hRSV-N RNA levels was cells into the airways of vehicle-treated mice (Fig. 4B, 4C). Fur-
observed at day 4 postinfection in the lungs of hRSV-infected ther, we analyzed the frequency of total leukocytes (CD45+) and
mice that were pretreated with CoPP (Fig. 3B). In contrast, neutrophils (MHC-II negative [MHC-II2] CD11c2 CD11b+ Ly-
mice receiving SnPP as pretreatment showed high viral loads in 6G+/Ly-6C+) in the BALF of hRSV-infected mice (Fig. 4B, 4C). A
the lungs (Fig. 3B). Furthermore, viral titers in BALF from mice significant decrease in the total cell counts of total leukocytes and
at 4 d postinfection were analyzed by immuno-plaque assays. neutrophils in BALF was observed for CoPP-pretreated mice upon
CoPP-treated mice showed a 2-log reduction in viral titers, as hRSV challenge, at day 4 postinfection (Fig. 4B, 4C). Contrarily,
compared with vehicle-treated mice (Fig. 3C). Further, qPCR SnPP-treated mice showed a higher amount of neutrophils in
analyses confirmed a significant increase in HO-1 mRNA levels in BALF at day 4 post hRSV infection (Fig. 4C). Additionally, hRSV
the lungs of CoPP-treated, hRSV-infected, and CoPP-uninfected infection increased the production of several cytokines and che-
mice as compared with control mice at day 4 postinfection mokines in BALF, which are associated with lung inflammation
(Supplemental Fig. 2A). These results suggest that the administra- (23). Thus, to evaluate whether the observed anti-inflammatory
tion of CoPP was effective at inducing HO-1 expression in the lungs effects of HO-1 in the course of hRSV infection were due to the
of mice. Furthermore, mRNA expression data were supported by modulation of cytokines or chemokines in BALF, protein levels of
flow cytometry analyses, which showed that CoPP treatment in- IL-6, IL-4, IFN-g, IL-10, CCL3/MIP-1a, and CXCL1/KC (an IL-8
creased the amount of HO-1 protein in epithelial Epcam-positive homolog) were measured by ELISA. Overall, the protein concen-
cells (Supplemental Fig. 2B). Therefore, our data suggest that trations of all hRSV-inducible cytokines (IL-6, IL-4, and IFN-g;
pharmacological induction of HO-1 expression, before virus chal- Fig. 5A–C) and chemokines (CCL3/MIP-1a and CXCL1/KC;

lenge, reduces viral replication and virus particle production. Fig. 5D, 5E) were lower in mice that were treated with CoPP, as
compared with vehicle-treated mice. Conversely, CoPP-treated mice
Induction of HO-1 expression reduces neutrophil infiltration
displayed increased secretion of the immunomodulatory cytokine
and lung inflammation during hRSV infection
IL-10, even in mock controls (Fig. 5F), as previously described (33).
To assess whether the pharmacological induction of HO-1 mod- Interestingly, SnPP-treated mice showed increased levels of CCL3/
ulates hRSV-mediated airway inflammation, histopathological MIP-1a (Fig. 5D), suggesting that the increase in neutrophil recruit-
analyses were performed in mice for each treatment at day 4 ment can be mediated by a higher concentration of this chemokine.
postinfection. Consistent with body weight loss and viral titration Taken together, these data suggest that the pharmacological
data, CoPP-treated mice displayed reduced inflammation after induction of HO-1 expression before virus challenge prevents the
hRSV infection, as compared to vehicle-treated control animals development of pulmonary inflammation by modulating the airway
(Fig. 4A, middle panel). Furthermore, CoPP-treated mice exhibi- immunological milieu during hRSV infection, as evidenced by a
ted reduced inflammatory infiltration in both bronchoalveolar reduction in lung inflammation and neutrophil infiltration in BALF.
airspaces and the lung interstitium, suggesting that HO-1 induc-
tion protects mice against bronchopneumonia and interstitial HO-1 induction increases antiviral type I IFN response during
pneumonia. Conversely, SnPP treatment caused a slight increase hRSV infection in vivo
in lung inflammation (Fig. 4A, bottom panel). BALF collected at Previous reports indicate that HO-1 can regulate early innate
day 4 postinfection and evaluated by flow cytometry for leukocyte immunity by modulating type I IFN production (14), as evidenced
FIGURE 3. HO-1 promotes disease resolution and viral clearance in mice experimentally infected with hRSV. BALB/cJ mice 6–8 wk old were treated
for 24 h with CoPP, SnPP, or vehicle control then inoculated intranasally either with mock or hRSV (1 3 106 PFU). hRSV disease progression was
monitored by (A) determining values for animal weight loss over 7 d [*p , 0.05, Student t test was applied between CoPP + hRSV (black open circle) and
hRSV (gray open triangle)]. (B) Lung homogenates of each experimental group of infected mice were collected at day 4 postinfection and quantified for
viral copy number assessing hRSV-N RNA per 5000 copies of b-actin of by qPCR. White bars represent the N RNA copy numbers for MOCK controls and
black bars represent hRSV infected mice. (C) The BALF from vehicle, CoPP-, and SnPP-treated mice were titrated on HEp-2 monolayers for the
quantification of infectious viral particles in the airways (expressed as PFUs per milliliter). Data shown are mean 6 SEM from three independent ex-
periments, each with three mice per group (n = 3). Data were analyzed by one-way ANOVA and multiple comparisons against the vehicle control were
performed for statistical analyses (*p , 0.05, **p , 0.001).

FIGURE 4. HO-1 induction reduces lung inflammation and inflammatory cell infiltration during hRSV infection. BALB/cJ mice 6–8 wk old were treated
for 24 h with CoPP, SnPP, or vehicle and then inoculated intranasally, either with mock or hRSV (1 3 106 PFU). Mice were euthanized and BALF analyzed
by flow cytometry. Lung sections were stained with H&E. (A) Histopathology analyses of lung sections from vehicle (upper panel), CoPP- (middle panel),
and SnPP-treated (bottom panel) and hRSV-infected mice, H&E, original magnification 310. (B) Representative dot plots of flow cytometry analysis for
vehicle, CoPP-, and SnPP-treated mice (upper panel) and absolute number cell count (bottom panel) of total CD45+ infiltration at day 4 postinfection in
BALF for the mentioned conditions. (C) Representative dot plots of flow cytometry analysis for vehicle, CoPP-, and SnPP-treated mice (upper panel) and
absolute number cell count (bottom panel) for neutrophil cell infiltration at day 4 postinfection in BALF (MHC-II2 CD11c2 CD11b+ Ly-6G/Ly-6C+) for
the mentioned conditions. Data shown are mean 6 SEM from three independent experiments, each with three mice per group. Values were analyzed by
one-way ANOVA and Bonferroni posttest (*p , 0.05, **p , 0.01, ***p , 0.0001).
by the inhibition of HCV replication after activation of this infected mice at day 4 postinfection. In addition, CoPP treat-
enzyme (34). Therefore, we evaluated whether CoPP-mediated ment also increased IFN-a/b mRNA expression in the lungs
HO-1 induction could activate an antiviral type I IFN response of mock (uninfected) control animals. Interestingly, SnPP
in vivo during hRSV infection. Expression of IFN-a/b was treatment failed to modulate this type I IFN response. These
measured in lungs from both hRSV-infected and control mice. data suggest that CoPP by itself induces an antiviral state in
As shown in Fig. 6, CoPP treatment enhanced mRNA levels of airway cells and that the upregulation of IFN-a/b requires HO-1
IFN-a (Fig. 6A) and IFN-b (Fig. 6B) in the lungs of hRSV- activity.
FIGURE 5. HO-1 inhibits hRSV-induced proinflammatory cytokine and chemokine responses in the airways of mice experimentally infected with hRSV.
BALF samples were obtained from mice treated with vehicle, CoPP, or SnPP to measure concentrations of proinflammatory cytokines (A) IL-6, (B) IL-4,
(C) IFN-g, (D) CCL3/MIP-1a, (E) CXCL1/KC (an IL-8 homolog), and (F) IL-10 by ELISA. Data shown are mean 6 SEM of three independent ex-
periments. Data were analyzed by one-way ANOVA and Bonferroni posttest (*p , 0.05, **p , 0.001, ***p , 0.0001).
FIGURE 6. HO-1 induction promotes the upregulation of type I IFNs. BALB/cJ mice 6–8 wk old were treated for 24 h with CoPP, SnPP, or vehicle then
inoculated intranasally, either with mock or hRSV (1 3 106 PFU). RNA from lungs of each experimental group was collected at day 4 and analyzed by
qPCR for IFN-a/b mRNA levels. (A) IFN-a relative mRNA expression levels for vehicle and CoPP or SnPP pharmacological-treated experimental groups.
(B) IFN-b relative mRNA expression levels for vehicle and CoPP or SnPP pharmacological-treated experimental groups. IFN-a/b relative expression was
normalized to b-actin levels and calculated using the 2-DDCt method (mock was used as a reference control). Data shown are mean 6 SEM from three
independent experiments, each with three mice per group (n = 3). Values were analyzed by one-way ANOVA and Bonferroni posttest (*p , 0.05, **p ,
0.01, ***p , 0.0001).
HO-1 induction slightly decreases T cell activation and controls and untreated transgenic mice (Fig. 8A). No significant
function during hRSV infection unspecific effects were observed for DOX treatment in uninfected

In addition to the important immunomodulatory properties of HO-1 mice (Fig. 8A). These results are consistent with the observations
on the innate immune response, this enzyme also exerts immu- described above, as at day 4 postinfection. We found a decreased
nomodulatory effects on T cell–mediated adaptive response, by neutrophil infiltration in the airways of tTA-HO-1 mice treated
impairing T cell activation, proliferation, and their effector func- with DOX, as compared with littermates treated with DOX and
tions (16, 35). To examine whether HO-1 induction affects T cell infected with hRSV (Fig. 8C). Furthermore, DOX-induced trans-
responses, single-cell suspensions were obtained from mediastinal genic mice also showed reduced viral loads in the lungs, as
lymph nodes of each experimental group and were stimulated for compared with infected littermates at day 4 postinfection with
72 h with ultraviolet-hRSV or with anti-CD3ε/CD28 (1 mg/ml), or hRSV (Fig. 8B). Finally, lung histological analyses were per-
left untreated. T cell activation in single-cell suspensions was formed to evaluate whether the transgenic expression of HO-1
determined by CD69 expression in CD4+ and CD8+ T cells as a could prevent lung inflammation. As shown in Fig. 8D, trans-
parameter for early activation (36). A significant increase in CD69 genic mice treated with DOX displayed significantly less inflam-
expression was observed in both CD4+ (Fig. 7A) and CD8+ mation than hRSV-infected littermate controls and untreated
(Fig. 7B) T cells stimulated with a polyclonal stimulus (anti- transgenic mice (Fig. 8D). Therefore, these data suggest that ex-
CD3ε/CD28) for all treatments. However, CoPP treatment led to a ogenous expression of human HO-1 in MHC-II+ cells decreases
reduction of CD69 expression on anti-CD3ε/CD28-stimulated neutrophil infiltration and lung inflammation in the conditional
cells (Fig. 7A, 7B). No significant changes were observed in tTA-HO-1 transgenic mice.
ultraviolet-hRSV stimulation, as evidenced by a non-significant Transgenic expression of human HO-1 in MHC-II+ cells
increase in CD69 expression, which had been previously de- modulates T cell function during hRSV infection
scribed by our group (36). In agreement with these data, a sig-
nificant amount of IFN-g was secreted in response to anti-CD3ε/ Next, we assessed whether exogenous expression of HO-1 in MHC-
CD28 stimulation with a mild decrease for the CoPP treatment II+ cells in conditional transgenic mice could affect the function of
(Fig. 7C). APCs and their ability to process and present Ags to T cells. First,
we evaluated the maturing capacity of DCs from tTA-HO-1. tTA-
Transgenic expression of human HO-1 in MHC-II+ cells HO-1 DCs were treated with vehicle, CoPP, or DOX for 2 h. Cells
modulates the pulmonary disease caused by hRSV were washed, and LPS (1 mg/ml) was added to the medium to
To confirm the HO-1 protective role against hRSV infection induce maturation (22). After 16 h, DC maturation was measured
through CoPP administration, conditional transgenic mice over- by surface expression of CD80, CD40, and CD86 molecules by
expressing the HMOX-1 gene in MHC-II+ cells (tTA-tHO-1) were flow cytometry. Overall, all assessed surface markers were up-
instilled intranasally either with hRSV (1 3 106 PFU) or mock. regulated in LPS-pulsed DCs, suggesting that HO-1 had no effect
Then 48 h before infection, 800 mg/ml DOX and 36 mg/ml in the maturation process of DCs in conditional transgenic mice
sucrose were added to drinking water to induce HO-1 expres- after HO-1 induction by DOX (Supplemental Fig. 4A). To eval-
sion in these transgenic mice. To address whether the treatment uate whether the ability of DCs to process and present Ags to
with DOX induces the expression of exogenous human HO-1 T cells could be altered by the transgenic expression of HO-1,
in MHC-II+ cells in the conditional transgenic mice tTA-tHO-1 OVA-pulsed mature DCs from tTA-HO-1 mice were cocultured
(Supplemental Fig. 3A), qPCR analyses of the human HO-1 gene with OT-II CD4+ T cells, which recognize the OVA-derived
were performed from lung tissue obtained from mice of each peptide OVA323–339/I-Ab complex as a cognate ligand. Thus,
experimental group (Supplemental Fig. 3B). The detection of LPS-DCs treated with CoPP showed an impairment in T cell ac-
human HO-1 in sorted pulmonary MHC-II+ cells further validated tivation after 48 h of coculture, suggesting that CoPP pretreated
this model (Supplemental Fig. 3C). Disease parameters were DCs were unable to prime OT-II T cells (Supplemental Fig. 4B).
evaluated as described above, in this transgenic mouse model On the contrary, mature DCs from the conditional transgenic mice
expressing human HO-1. Consistent with the effects observed in that were treated with DOX were capable of processing and pre-
CoPP treatment, the selective expression of human HO-1 induced senting OVA-derived peptides, resulting in OT-II T cell priming.
by DOX resulted in an accelerated kinetic of body weight re- However, the activation of OT-II T cells in the coculture with DCs
covery at days 3 and 4 postinfection, as compared with littermate from conditional transgenic mice treated with DOX was lower
FIGURE 7. HO-1 induction slightly decreases T cell response during hRSV infection. Single-cell suspensions were obtained from mediastinal lymph
nodes to evaluate T cell response in infected mice treated with vehicle, CoPP, or SnPP. The collected cells were stimulated with ultraviolet-inactivated
hRSV or anti-CD3ε/CD28, or left unstimulated for 72 h. Flow cytometry detection of CD69 expression by CD4+ (A) and CD8+ (B) T cells derived from
each group of mice treated without stimuli, or with hRSV or anti-CD3ε/CD28 Abs. (C) IFN-g secretion detected by ELISA in the supernatant of lymph
nodes at 72 h poststimulation with hRSV-or anti-CD3ε/CD28. Data shown are mean 6 SEM from three independent experiments.
compared with cocultures between vehicle-treated DCs and OT-II Discussion

cells (Supplemental Fig. 4B), suggesting that the expression of HO-1 is a stress-inducible enzyme that catalyzes heme degradation
HO-1 in DCs from conditional transgenic mice slightly affects the into carbon monoxide, biliverdin, and free iron (37). HO-1 in-

T cell priming. Furthermore, to assess whether expression of HO- duction has been involved in several pathophysiological condi-
1 in MHC-II+ cells affects T cells priming in vivo during hRSV tions, and HO-1 is generally regarded as a cytoprotective enzyme
infection, littermate, untreated or DOX-treated tTA-tHO-1 mice (38–40). Recently, there has been a growing interest about the
were infected with hRSV and 4 d postinfection, single-cell sus- regulatory role that HO-1 may play in immunity and tolerance (41,
pensions were obtained from mediastinal lymph nodes and stim- 42). Several studies support the beneficial effects that HO-1 and its
ulated for 72 h with ultraviolet-hRSV or with anti-CD3ε/CD28 products can have during inflammatory processes (42–44). How-
(1 mg/ml) or left untreated. To evaluate T cell activation in the ever, little is known about the contribution of HO-1 expression
cells suspensions, CD69 expression was measured in CD4+ during viral infections in vivo. Previous evidence described that
T cells. No significant increases in CD4 or CD69 expression were gene overexpression of HO-1 can attenuate disease severity of
observed for cell suspensions from anti-CD3ε/CD28-stimulated influenza infection in mice (45). Moreover, induction of HO-1
DOX-induced transgenic mice as compared with unstimulated inhibited influenza replication by activating the type I IFN sys-
DOX-induced transgenic mice, suggesting that T cell activation tem with the subsequent induction of IFN-stimulated genes (46).
is affected by HO-1 overexpression in MHC-II+ cells (Fig. 9A). Further, HO-1–deficient (ho-12/2) mice displayed decreased sur-
In agreement with these data, no significant IFN-g secretion vival rates after influenza infection, as compared with wild-type
could be measured in supernatants of cell suspensions derived mice (47).
from DOX-induced transgenic mice (Fig. 9B), suggesting that In this study, we evaluated whether the induction (or inhibition)
T cell function can also be modulated by HO-1 overexpression in of HO-1 could affect the development of the disease caused by
MHC-II+ cells. hRSV in mice. Our findings indicate that preadministration of CoPP
FIGURE 8. HO-1 overexpression in the MHC-II+ cell subset reduces disease symptoms in rtTA-HO-1 hRSV-infected mice. DOX and sucrose were added
to the drinking water of littermate or conditional transgenic mice (tTA-HO-1). (A) Mice were inoculated intranasally, either with hRSV or mock (non-
infected supernatant) and hRSV disease progression was monitored by analyzing animal weight loss during 4 d (*p , 0.05, Student t test between LM DOX
hRSV + tTA-HO-1 DOX hRSV values). (B) Lung homogenates of each experimental group of mice were collected at day 4 postinfection, and quantified for
viral RNA by qPCR, using primers targeting the hRSV-N gene. Data in the graph show N-RNA copy numbers per 5000 copies of b-actin. (C) Neutrophil
absolute cell count infiltration (MHC2II2 CD11c2 CD11b+ Ly-6G/Ly-6C+) at day 4 in BALF for mock and hRSV-infected mice for each experimental
group. (D) Histopathology analyses of lung sections for each experimental group (H&E, original magnification 310). Data shown are from two inde-
pendent experiments, each with three mice per group (n = 2). Mann–Whitney U test (*p , 0.05, **p , 0.01).
FIGURE 9. Exogenous HO-1 expression in MHC-II+ cells of conditional transgenic mice impairs T-cell function during hRSV infection. DOX and
sucrose were added to the drinking water of transgenic mice (tTA-HO-1) infected with hRSV or mock (non-infected supernatant). LM mice were included
as a control. Single-cell suspensions were obtained from mediastinal lymph nodes to evaluate T cell response, in the conditional transgenic mice, to hRSV
infection. The collected cells were stimulated with ultraviolet-inactivated hRSV or anti-CD3ε/CD28 or left unstimulated for 72 h. (A) Flow cytometry
detection of CD69 expression on CD4+ T cells derived from mice, treated without stimuli, or with hRSV or anti-CD3ε/CD28 Abs. (B) IFN-g secretion
detected by ELISA in the supernatant of lymph nodes at 72 h poststimulation with hRSV or anti-CD3ε/CD28. Data shown are mean 6 SEM from three
independent experiments.

can promote a significantly faster body weight recovery after hRSV lung tissue, leading to the secretion of proinflammatory media-
infection, as compared with control mice. Similar results were tors (49, 50). Because HO-1 has been found to inhibit NF-kB
observed in transgenic tTA-HO-1 mice that overexpress human activity (51), we hypothesized that HO-1 induction decreases
HO-1 in MHC-II+ cells. However, inhibition of HO-1 activity lung inflammation through the inhibition of NF-kB, which re-
mediated by SnPP preadministration did not increase body weight sults in lower levels of proinflammatory cytokines and chemo-
loss as a parameter of disease, suggesting that HO-1 enzymatic kines. Furthermore, HO-1 induction increases IL-10 secretion in
activity is not necessarily the unique element involved in the BALF, supporting an anti-inflammatory role for HO-1 during
protective effect observed in the body weight recovery induced by hRSV infection.
CoPP. In addition, we found that CoPP preadministration reduced Consistent with the observations described in this study for
viral mRNA and the titers of infective viral particles in the lungs hRSV, decreased neutrophil infiltration and lung injury were de-
of hRSV-infected mice, an outcome that coincided with an in- scribed during influenza virus H1N1 infection as a result of
crease in HO-1 expression in lungs of mice at day 4 postinfection adenoviral-mediated HO-1 gene transfer (45). These findings
(Supplemental Fig. 2). Interestingly, we observed that SnPP pre- support the notion that HO-1 activity may be important for
treatment caused a slight increase in viral loads in the lungs during modulating lung inflammation during viral pulmonary patholo-
the same period of time, however, viral progeny (PFU) was not gies (47, 52). This idea was further supported by the observation
raised. These data suggest that the HO-1 enzymatic activity or its that mice deficient for Nrf2 (nrf2 2/2), a transcription factor
products are not necessarily involved in the modulation of viral controlling HO-1 gene expression, displayed a significantly re-
replication and clearance. The increased expression of HO-1, duced hRSV clearance, a higher bronchopulmonary inflamma-
observed in Epcam-positive cells from the lungs of CoPP- tion, and a reduced body weight gain as compared with control
pretreated mice, is consistent with the notion that a reduction in mice (53).
viral loads could be related to the promotion of an antiviral re- The results observed for pharmacological induction of HO-1
sponse, mediated by HO-1 in airway epithelial cells. This concept mediated by CoPP are similar. The conditional expression of
is supported by the observation that although CoPP reduced viral HO-1 in MHC-II+ subsets in vivo promoted a reduction in lung
loads and virus particle production in hRSV-infected A549 cells, disease in hRSV-infected tTA-HO-1 transgenic mice. These data
SnPP-mediated inhibition of HO-1 activity increased both pa- suggest that the HO-1 enzyme is responsible for the protective
rameters. However, the initial steps of the life cycle of the virus, effect of CoPP treatment. Importantly, although the MHC-II
such as hRSV binding and entry, were not affected by HO-1 in- molecule is constitutively expressed by APCs, its expression is
duction. Furthermore, we observed that CoPP treatment induced not limited to immune cells. In fact, intestinal and pulmonary
an upregulation of IFN-a/b in the lungs of hRSV-infected mice, epithelial cells can also express this molecule (54). Then, the
indicating that HO-1 plays an important role during the develop- conditional transgenic model used in this work suggests that
ment of the antiviral type I IFN response in the airways. All these the upregulation of HO-1 in epithelial cells and immune cells
data suggest that HO-1 induction could be promoting an antiviral expressing MHC-II can contribute to suppressing the inflamma-
state that prevents or limits viral replication and propagation of tion triggered by the hRSV infection.
new virus particles. It is important to mention that HO-1 was highly induced in DCs
In contrast, during acute lung infections, the host inflamma- exposed to hRSV and it has been described that infection of DCs
tory response requires tight regulation (48) to promote pathogen by hRSV impairs the ability of prime T cells (55), suggesting that
clearance without evoking an exaggerated inflammatory response HO-1 induction in response to hRSV could contribute to this
that could damage the infected airways. Our results indicate that process. Furthermore, HO-1 upregulation mediated by CoPP treat-
HO-1 upregulation results in the suppression of lung inflamma- ment or the exogenous expression of human HO-1 in MHC-II+
tion, associated with a decrease in the inflammatory cell infiltra- cells in the conditional transgenic model slightly affects T cell
tion and an inhibition of proinflammatory cytokine or chemokine activation and function, indicating that Ag processing and presen-
secretion during hRSV infection. Several studies have reported tation by DCs was impaired by HO-1, in agreement with previous
that hRSV infection actively induces activation of NF-kB in the data supporting a role of HO-1 during adaptive immunity (35).
Treatment based in HO-1 upregulation is currently used as a 15. Shan, Y., R. W. Lambrecht, S. E. Donohue, and H. L. Bonkovsky. 2006. Role of
Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt pro-
therapeutic approach. The systemic hemin therapy, an HO-1 in- toporphyrin. FASEB J. 20: 2651–2653.
ducer, has been approved by the Food and Drug Administration to 16. Espinoza, J. A., P. A. González, and A. M. Kalergis. Modulation of antiviral
treat acute intermittent porphyria (56). Hemin also has been used immunity by heme oxygenase-1. Am. J. Pathol. 187: 487–493.
17. Ewing, P., A. Wilke, G. Eissner, E. Holler, R. Andreesen, and A. Gerbitz. 2005.
to treat thalassemia intermedia, myelodysplastic syndrome, and Expression of heme oxygenase-1 protects endothelial cells from irradiation-
liver allograft failure in erythropoietic protoporphyria (56). How- induced apoptosis. Endothelium 12: 113–119.
ever, this strategy has not been explored for the treatment of in- 18. Sardana, M. K., and A. Kappas. 1987. Dual control mechanism for heme oxy-
genase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly
fectious diseases. increasing content of enzyme protein in liver. Proc. Natl. Acad. Sci. USA 84:
In summary, the current study demonstrates that hRSV infec- 2464–2468.
tion can be modulated by the expression of HO-1 both in vitro 19. Witherden, D., N. van Oers, C. Waltzinger, A. Weiss, C. Benoist, and D. Mathis.
2000. Tetracycline-controllable selection of CD4(+) T cells: half-life and sur-
and in vivo. Thus, HO-1 activity may play a critical role during vival signals in the absence of major histocompatibility complex class II mol-
hRSV infection. HO-1 induction could protect the host from the ecules. J. Exp. Med. 191: 355–364.
pulmonary pathology developed upon hRSV infection, by reducing 20. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu,
and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from
viral replication and lung inflammation, thus favoring disease mouse bone marrow cultures supplemented with granulocyte/macrophage
resolution. Therefore, our results shed light on the potential role of colony-stimulating factor. J. Exp. Med. 176: 1693–1702.
the therapeutic induction of HO-1 in this viral pneumonia and 21. Bueno, S. M., P. A. González, K. M. Cautivo, J. E. Mora, E. D. Leiva,
H. E. Tobar, G. J. Fennelly, E. A. Eugenin, W. R. Jacobs, Jr., C. A. Riedel, and
suggest new avenues for the immunomodulatory treatment of A. M. Kalergis. 2008. Protective T cell immunity against respiratory syncytial
hRSV-infected patients. virus is efficiently induced by recombinant BCG. Proc. Natl. Acad. Sci. USA
105: 20822–20827.
22. Riquelme, S. A., S. M. Bueno, and A. M. Kalergis. 2015. Carbon monoxide
Acknowledgments

down-modulates Toll-like receptor 4/MD2 expression on innate immune cells
We thank Dr. Pedro Piedra (Baylor College of Medicine), Dr. George and reduces endotoxic shock susceptibility. Immunology 144: 321–332.
23. Jafri, H. S., S. Chávez-Bueno, A. Mejı́as, A. M. Gómez, A. M. Rı́os, S. S. Nassi,
Kollias, and Dr. Christophe Benoist for kindly providing the A549 cells, M. Yusuf, P. Kapur, R. D. Hardy, J. Hatfield, et al. 2004. Respiratory syncytial
pIi-TTA-TetO-HO-1, and pIi-TTA transgenic mice, respectively. We also virus induces pneumonia, cytokine response, airway obstruction, and chronic
thank Marı́a José Altamirano for breeding the mouse colonies that were inflammatory infiltrates associated with long-term airway hyperresponsiveness
used in this work. in mice. J. Infect. Dis. 189: 1856–1865.
24. Bunse, C. E., V. Fortmeier, S. Tischer, E. Zilian, C. Figueiredo, T. Witte,
R. Blasczyk, S. Immenschuh, and B. Eiz-Vesper. 2015. Modulation of heme
Disclosures oxygenase-1 by metalloporphyrins increases anti-viral T cell responses. Clin.
Exp. Immunol. 179: 265–276.
The authors have no financial conflicts of interest. 25. Battles, M. B., J. P. Langedijk, P. Furmanova-Hollenstein, S. Chaiwatpongsakorn,
H. M. Costello, L. Kwanten, L. Vranckx, P. Vink, S. Jaensch, T. H. Jonckers, et al.
2016. Molecular mechanism of respiratory syncytial virus fusion inhibitors. Nat.
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Supplementary Figure 1. CoPP-mediated HO-1 induction in epithelial cells. A549 cells
were infected with hRSV in the presence or absence of CoPP or SnPP treatment. Also, mock
and UV-hRSV treatments were included as experimental controls. Then, cells were analyzed
by flow cytometry for HO-1 expression, which is represented as geometric mean
fluorescence intensity (GMFI). White bars represent pre-infection treatment with drugs and
black bars indicate drug treatment post-infection. Data were analyzed by One-way ANOVA
and the Bonferroni post-test (*, P< 0.05; **, P< 0.001, ***, P< 0.0001). Data shown are
means +/- SEM from three independent experiments.
Supplementary Figure 2. CoPP-treatment induces HO-1 expression on epithelial cells
in lung mice. 6-8 week old BALB/cJ mice were treated for 24 h with CoPP, SnPP or control
vehicle and then intranasally inoculated with either mock or hRSV (1x106 PFU). (A) Lung
homogenates of each experimental group of mice (vehicle, CoPP or SnPP-treated and hRSV-
infected mice) were collected at day 4 post-infection and HO-1 relative mRNA expression
levels were evaluated (***, P< 0.001 One-way ANOVA). Relative levels of mRNA HO-1
were normalized to β-actin levels by using the 2-ΔΔCt method (vehicle treated mice were
used as a reference control). (B) Whole lung cell suspension was analyzed by flow cytometry
for HO-1 expression at day 4 post-infection. Representative dot plots of flow cytometry
analyses, showing gating strategy to separate Epcam epithelial cells and CD45 immune cells.
Then, representative histograms show expression of HO-1 enzyme in each population. (C)
MFI of HO-1 in whole lung cell suspensions of Epcam and CD45 cell populations. Values
were analyzed by One-way ANOVA and the Bonferroni post-test (*, P< 0.05, **, P< 0.01).
Data shown are means +/- SEM from three independent experiments.
Supplementary Figure 3. Human doxycycline-induced HO-1 expression is limited to
MHC-II+ cells in conditional transgenic mice tTA-HO-1. (A) rtTA strain expresses the
tetracycline-controlled transactivator protein (tTA) under the MHC-II Eα promoter that
controls the expression of tTA in MHC-II+ cells. When these transgenic mice are mated to a
second transgenic strain carrying a gene of interest (HMOX-1 or human HO-1) coupled to a
tetracycline-responsive promoter element (TRE), conditional expression of the target gene in
MHC-II+ cells may be controlled by the administration of tetracycline or doxycycline. (B)
Lung homogenates of conditional transgenic tTA-HO-1 and littermate mice with or without
administration of DOX were obtained to obtain mRNA and relative expression of human
HO-1 were measured. (C) Single-cell suspensions were obtained from spleen of conditional
transgenic mice that receive DOX in drinking water for 24 h. Then, cells suspensions were
separated by MACs in a negative MHC-II population (right upper panel) and MHC-II-
enriched (left upper panel). Later, the percentage of HO-1 expression was measured in each
population (bottom panel). Data shown are means +/- SEM from two independent
experiments.
Supplementary Figure 4. DCs from tTA-HO-1 conditional transgenic mice are
impaired for antigen processing/presentation and a normal maturation process. tTA-
HO-1 DCs derived from bone marrow progenitors were stimulated with LPS for 16h in
presence or absence of CoPP, DOX or vehicle. (A) Flow cytometry analyses of surface
expression of the maturation markers CD40, CD80, and CD86 in LPS-stimulated or
unstimulated DCs. Data were expressed as MFI. (B) tTA-HO-1 DCs were stimulated with
OVA in presence or absence of CoPP, DOX or vehicle 24 h. Next, OVA-DCs were co-
cultured with OT-II T cells for 48 h and surface expression of CD69 was evaluated by flow
cytometry. Results in graph are the percentages of CD4/CD69 positive cells. Data shown are
means +/- SEM from two independent experiments.

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